Method for identifying a compound that modulates the function of the gene product of an essential gene

ABSTRACT

A method for identifying a compound which modulates the function of the gene product of an essential gene, which method comprises providing viable cells wherein the gene is expressed under the control of a heterogenous, regulatable promoter, switching off gene expression via the promoter, contacting the cells with a test compound and determining any modulatory effect of the compound on the function of the gene product.

This application is the National Phase of International ApplicationPCT/GB00/02472 filed Jun. 27, 2000 which designated the U.S. and thatInternational Application was published under PCT Article 21(2) inEnglish.

This invention provides methods for the identification of novelbiological targets and/or inhibitors. Such methods provide useful andconvenient tools for high throughput screening. In particular, suchmethods may be used to identify novel antimicrobial agents.

The emergence of resistance to current therapeutic agents, such asantibacterial, antifungal, and antimalarial agents, necessitates thedevelopment of novel agents. Whilst target-based biochemical screens canbe successful, classic anti-microbial screening has clearly outperformedrational target-selecting procedures in terms of discovered novelantimicrobials. Target-based biochemical screens are frequently seen toinvoke a lack of cell activity, despite a good inhibition of the targetenzyme, perhaps due to low permeation into or export out of the cell.Also the presence of inhibiting structures for a hand-picked targetwithin a limited set of available molecules is by no way guaranteed.

In contrast, cell-based antimicrobial screening frequently identifiesantimicrobial action but then either reveals this inhibition to becaused by general toxicity, for example membrane perturbation orDNA-intercalation, or fails to identify any specific interaction of thedrug with the cellular machinery. This complicates chemical optimisationof initial leads, providing no guide for a structure-activityrelationship.

A particular approach to identify target-based mode of action has beenthat of hypersensitivity.

Historically, the way to generate hypersensitive versions of a proteinof interest has been the generation of temperature-sensitive (ts)mutations in its encoding gene (Schmid et al. Genetics 123, pp625-633,(1989)). Temperature sensitive (Ts) or other conditional phenotypes canbe generated by either targeted (for example using PCR) or random (forexample using chemicals or radiation) mutagenesis. However, suchconditional mutants are frequently hypersensitive to inhibitors evenunder permissive conditions.

We have now found that hypersensitivity to inhibitors can be readilyachieved by shutting down a particular essential function of viablecells followed by analysis of the effects in the presence/absence of apotential inhibitor. By “shutting down” we mean that the particularessential function is not available to the cells at any expressionlevel.

Therefore in a first aspect of the present invention we provide a methodfor identifying a compound which modulates the function of the geneproduct of an essential gene, which method comprises providing viablecells wherein the gene is expressed under the control of aheterogeneous, regulatable promoter, switching off gene expression viathe promoter, contacting the cells with a test compound and determiningany modulatory effect on the function of the gene product.

The method provides a step jump in the feasibility of a complete genomeanalysis for compound hypersensitivity. It provides a number ofsignificant advantages not least that the unaltered “normal” protein isused in contrast to for example extrapolation from the interaction of analtered, mutagenized protein with an inhibitor onto the interaction ofthe same chemical. Also no effort is needed to fine tune promoteractivity to a higher or lower level. Inhibition of one specificbiochemical entity, for example a protein required for cellular growthor survival is conveniently indicated by a hypersensitive response ofthe strain with the regulatable gene compared to its unaltered parent.

By “heterogeneous, regulatable promoter” we mean a promoter other thanthe native promoter for the essential gene and which can be regulated bythe addition and/or removal of specific materials or for example byother environmental changes.

By “hypersensitivity” we mean a larger reduction in cell growth in thepresence of an identical concentration of drug, compared to a wild-typecell.

Furthermore, hypersensitivity caused by underexpression of one specificenzyme can extend to a whole biochemical pathway, such as for examplesterol biosynthesis. This is exemplified by the results shown in FIG. 5of this invention, where the sensitivity to terbinafine is altered bothby shutdown of the target enzyme (encoded by ERG1) as well as shutdownof another enzyme (encoded by ERG11) in the same pathway.

By “viable” we mean cells that have grown sufficiently so that when geneexpression of an essential gene is turned off, meaningful measurementsand kinetic analysis may be made. The cells are preferably allowed togrow into early stationary phase by which time a final optical densityreading is taken. A reduced optical density compared to no drug or noswitchoff controls is interpreted as growth inhibition.

By “essential gene” we mean a gene required by a cell for example forcell growth and/or cell viability. This does include genes required onlyunder certain assayable conditions (ie conditional essential). In itssimplest manifestation essentiality is defined as necessary for growthof the organism on rich media (For a comprehensive summary of such mediaas well as general yeast methodology see Sherman et al in Methods inEnzymology, Vol 194, Guthrie and Fink eds, Academic Press (1991).Convenient genes include fungal and bacterial genes.

By “switching off gene expression” we mean that all gene expression isturned from ON to OFF. There is no low level gene expression in the OFFposition.

By “cells” we mean cells from any convenient source, these includehuman, animal or microbial cells. New genome-based techniques aredeveloping which combine screening of compounds with simultaneous targetidentification. Whilst this invention is of particular use withgenetically tractable model organisms such as Schizosaccharomyces pombeand Saccharomyces cerevisiae, it is universally applicable and islimited only by practical considerations. Using model organisms such asS. cerevisiae, S. pombe, C. elegans, or D. melanogaster it is possibleto study conserved functions of eukaryotes. The application of geneticsusing human or animal cell lines directly, then extends possibilitiesfurther.

Any convenient test compound such as a peptide, nucleic acid and lowmolecular weight compound, may be used in the methods of the invention.Preferred test compounds are potential therapeutic agents or may be usedin further studies to identify therapeutic agents. Particular testcompounds are low molecular weight compounds of, for example, molecularweight of less than 1000, such as less than molecular weight 600.

The modulatory effect of a compound on the gene product of an essentialgene is conveniently investigated taking endpoint optical densityreadings (the higher the absolute optical density of the culture, theless inhibition is thought to have occured. Such analysis isconveniently effected over period of typically 24 hours for yeast cells.Bacteria may take significantly shorter (eg 12 hours) whereas highereukaryotic cells will require several days to reach early stationarygrowth phase. The analysis is conveniently photometric (optical densityof the culture at 600 nm wavelength).

Importantly, the combination of very recent techniques for site specificintegration of DNA (for example with a switchable promoter directlyreplacing the native one) with the observed fixed timepoint opticaldensity analysis of the hypersensitive response (as further elaboratedbelow) is novel and allows exploitation for large-scale drug screening,compound and target profiling. Moreover, such strains are also veryeasily obtainable in very high quantity (ie. thousands of genes in S.cerevisiae).

Some examples of switchable promoters for use in S. cerevisiae includeMET3 (repressible by added methionine) and GAL1 (repressed by glucoseinduced by galactose); for use in S. pombe: NMT1 (repressed bythiamine); for use in C. albicans: MAL1 (repressed by glucose, inducedby maltose, sucrose); for use in E. coli: araB (repressed by glucose,induced by arabinose); for use in Gram-positive bacteria such asStaphylococci, Enterococci, Streptococci and Bacilli: xylA/xylR (from S.xylosus) (repressed by glucose, induced by xylose); for use in E.coliand B.subtilis pSPAC (an artificial promoter derived from E.coli lac,regulated by IPTG, see Vagner et al. Microbiology (1998), 144,3097-3104); and for all of the above organisms plus further unspecifiedfungi, bacteria and mammalian cell lines: tetA/tetR (from variousbacterial tetracycline resistance cassettes) this system exists invarious versions, see Gossen et al. Current Opin. Biotechnol. 5,pp516-520 (1994), that are repressible or inducible by varioustetracycline analogues.

We illustrate in the Example and Figures below that it is possible togenerate hypersensitivity towards any agent that inhibits a biochemicalfunction by placing the gene encoding that function (protein or RNA)under control of a tightly regulatable promoter and switching it fromthe ON to the OFF state whilst simultaneously adding the interactingsubstance (for illustration of such a switch see FIG. 1). Whilst it isknown to place external promoters in front of genes of interest toachieve over- or under-expression of the encoded protein, this processrequires labour-intensive fine-tuning and setting up. Due to stronglyvarying expression levels, every gene of interest would require adifferent tuning of its promoter strength to be at a justgrowth-rate-limiting level. Therefore such a protocol relying onfine-tuning is unlikely to allow implementation at the high throughputscale necessary for genomic analysis.

Our novel findings, as detailed in the experiments shown below,demonstrate that a simple and standard protocol using just one promoterat clearly defined ON and OFF states (ie. explicitly without any finermodulation of the expression level) is capable to generate accuratemeasurements of specific hypersensitivity due to target underexpression.The underexpression is caused by i) decay of the RNA, ii) decay of thetarget protein, iii) dilution of the target molecule into severaldaughter cells during cell growth in the absence of any resynthesis.

The examples below demonstrate, for example, that unknown fungal targetproteins that can be inhibited by compounds can be identified by placingtheir essential genes under control of a regulatable promoter, switchingit off and exposing it to compound levels that do not inhibit thecorresponding wild type cell. Currently 814 S. cerevisiae genes havebeen shown essential for growth even on rich media, (source YPD™.) Anobserved hypersensitive response (eg in a fungus such as S. cerevisiae)shows: i) the target is essential ii) the compound is an antifungalagent or a starting point for evolution of a more potent analogue iii)the matching target-compound pair (or pairs) establishes mode of actionfor the antifungal compound. For more than one compound detected againstone target, valuable structure-activity relationship (SAR) informationcan be obtained. Comparison and pattern recognition analysis of manycompound effects on many targets will establish a valuable databaseuseful for grouping novel targets into pathways immediately.Hypersensitivity also allows the target-specific detection of lowpotency compounds not identifiable using the parental strain.

A further advantage of the invention is that characterised biochemicalactivity of the target molecule is not a pre-requisite for the methodsof the invention. Hypersensitivity as such can provide an assayablefeature intrinsically linked with a specific target. Genomic switchoffconstructs can be generated and integrated into the organisms genome invery high throughput using published PCR-based methodology without theinvolvement of any cloning step (Longtine et al. Yeast, 14, pp 953-961(1998); Zhang et al. Nature Genetics, 20, pp 123-128 (1998). Thereforethe methods of the invention may be used to test as many compounds aspossible against every potential antifungal target, for example up to10, up to 20, up to 50, up to 100, up to 500, or up to 1000 targetsusing modern high throughput screening technology.

Therefore in a further method of the invention we provide a method foridentifying metabolic pathway drug hypersensitivity which methodcomprises providing viable cells wherein a gene in the metabolic pathwayis expressed under the control of a heterogenous, regulatable promoter,switching off expression of the gene via the promoter, contactingdifferent groups of the cells with different test compounds, determiningand comparing any modulatory effect on the growth of the viable cells.Underexpression of one element of a biochemical pathway and chemicalinhibition of another part has been shown to be synergistic (see FIG. 5)thus generating pathway specific information by underexpressing just oneelement of that pathway.

The invention will now be illustrated but not limited by reference tothe following Example and Figures wherein:

FIG. 1 shows the generation of a controllable allele of gene, Gen1, byreplacing its native promoter with a well defined and tight ON/OFFswitch; here GAL1

FIG. 2 shows Erg11p activity in wild type (wt) and GAL1-ERG11 strainsand illustrates how catalytic activity declines both with time andincreased concentration of an inhibitor. The culture is shifted fromgalactose to glucose at time 0, whereby new synthesis of Erg11p(lanosterol C-14-demethylase) is stopped. The fluconazole concentrationis sub-inhibitory (for example 10 mg/l) for a wild type (wt)strain.;_(—) _(—:)no fluconazole, . . . : fluconazole; the threshold iswhere Erg11p activity is rate-limiting for growth.

FIG. 3 shows the growth of wild type (JK9-3da, Kunz et al. Cell, 73,pp585-596(1993)), GAL1-ERG11 and GAL1-AUR1 (generated as described inLongtine et al. Yeast, 14, pp 953-961 (1998))strains in the presence offluconazole;

FIG. 4 shows the growth of wt, GAL1-ERG11, and GAL1-AUR1 strains in thepresence of aureobasidinA;

FIG. 5 shows the growth of wt, GAL1-ERG11, and GAL1-AUR1 strains in thepresence of terbinafine;

FIG. 6 shows a protocol for promoter replacement in S. cerevisiae(substantially as described in Longtine et al. Yeast, 14, pp 953-961(1998)).

EXAMPLE The Ergosterol and Sphingolipid Biosynthetic Pathways of S.cerevisiae: a Study With Known Inhibitors

The rationale in using a complete switchoff instead of a low expressoris detailed as follows exemplified for Erg11p (p=protein), thelanosterol C-14-demethylase, a fungal enzyme in the ergosterolbiosynthetic pathway: The drug fluconazole, like other azoleantifungals, inhibits Erg11p leading to a decreased total activitywithin the cell. However no reduction in growth will be observed as longas the amount of active Erg11p is high enough not to be rate limitingfor growth (above the threshold, see FIG. 2) In our example thefluconazole concentration is insufficient to reduce Erg11p activitybelow this threshold. The situation for a GAL1-driven ERG11 gene isdifferent however: At time 0 the gene's engineered promoter is switchedfrom the ON state (because of the high expression of a GAL1 promoterthis level is usually higher than for the native promoter) to the OFFstate by changing the carbon source in the culture. From this timepointon no new mRNA is transcribed and—after decay of the mRNA—no new Erg11protein is being synthesized. Erg11 concentrations are now falling dueto the dilution of the enzyme into new daughter cells and protein decayof Erg11p. As soon as the threshold line is crossed (FIG. 2) Erg11pbecomes rate-limiting ie the strain shows a reduced growth rate comparedto the unaltered parental strain (wt). This is reflected by a lowerfinal optical density of the culture. The presence of the inhibitorfluconazole titrates active Erg11p molecules rendering them inactive.Therefore the threshold-line is crossed earlier, as no new Erg11p can besynthesized. Compared to the wild-type cell, this is seen ashypersensitivity (larger reduction in growth in the presence of anidentical concentration of drug). Simplified one can explain this as areduced amount of cell-doublings before rate-limiting conditions occur.

For target and compound evaluation the hypersensitivity has to bespecific for certain drug-target combinations. Therfore the method wastested and validated with another unrelated known drug-targetcombination: The AUR1 gene encodes inositolphosphorylceramide (IPC)synthase, an essential enzyme of the fungal sphingolipid biosyntheticpathway (Nagiec et al, J.Biol.Chem., 1997, 272,15, 9809-9817). Theenzyme is inhibited by the natural product aureobasidinA with subnanomolar potency, leading to an antifungal action of the drug. LikeERG11, AUR1 was subjected to a promoter swap and the resulting yeaststrains were the parent (wt), GAL1-ERG11 and GAL1-AUR1. They were testedfor their sensitivity towards both drugs, fluconazole and aureobasidinA.As predicted by our theory both GAL1-driven strains showed a prominenthypersensitive response towards their cognate drugs (see FIGS. 3 and 4).Remarkably, there was no cross reaction ie GAL1-ERG11 showed noincreased sensitivity towards aureobasidinA, neither did GAL1-AUR1 showhypersensitivity to fluconazole. This result confirms our theory andproves applicability and utility of the described method as a drug andtarget discovery tool.

It follows that the hypersensitive reaction towards an inhibitor of oneenzyme in a pathway may be extended to inhibitors of the whole pathway.This is due to the linear arrangement of enzymes in a biochemicalcascade of reactions where one enzyme provides substrate for the next. Areduced substrate supply can act synergistically with a reduced enzymelevel. Thus the activity of the almost rate-limiting enzyme Erg11p(after switchoff) will be reduced further by withdrawal of its substratenormally provided by Erg1p, squalene epoxidase, another enzyme of thesterol biosynthetic pathway, through several biochemical steps. Erg1p isnow inhibited by terbinafine. Any such pathway specific hypersensitivityallows the categorisation of (uncategorised) genes into the same pathwayas the switched off gene. To investigate this, we examined Erg1p. Thisenzyme has been shown to be inhibited by the antifungal terbinafine butErg11p is not. As shown in FIG. 5, GAL1-ERG11 is as hypersuscepible toterbinafine as GAL1-ERG1, proving the postulated cross-pathwayhypersensitivity. This pathway-specific analysis tool will be extremelyuseful for the classification of novel inhibitors (with unknown mode ofaction).

It will be appreciated that whilst exemplified particularly for S.cerevisiae, this invention is universally applicable in all geneticallymanipulatable organisms such as bacteria.

What is claimed is:
 1. A method for identifying a compound whichmodulates the function of the gene product of a gene essential for cellgrowth and/or cell viability under assayable conditions, which methodcomprises: (a) providing viable cells wherein the gene is expressedunder the control of a heterogenous, regulatable promoter; (b) switchingoff expression via the promoter; (c) contacting the cells with a testcompound; and (d) determining a modulatory effect on the function of thegene product.
 2. A method as claimed in claim 1, wherein the viablecells in step (a) have grown into early stationary phase.
 3. A method asclaimed in claim 1, wherein the gene is a bacterial gene.
 4. A method asclaimed in claim 1, wherein the gene is a fungal gene.
 5. A method asclaimed in claim 1, wherein the modulatory effect is determined viaoptical density measurements.
 6. A drug screening method which comprisesusing a method as claimed in claim 1 to test a plurality of testcompounds against gene products of more than one essential gene.
 7. Amethod as claimed in claim 6, wherein the compounds are tested againstthe gene products of more than 20 essential genes.
 8. A method foridentifying metabolic pathway drug hypersensitivity, which methodcomprises: providing viable cells wherein a gene in the metabolicpathway is expressed under the control of a heterogenous, regulatablepromoter; switching off expression of the gene via the promoter;contacting a first population of the cells with a first test compound;contacting a second population of the cells with a second test compounddifferent from the first test compound; and determining and comparing amodulatory effect of the first and second test compounds on the growthof the cells.
 9. A method as claimed in claim 2, wherein the gene is abacterial gene.
 10. A method as claimed in claim 2, wherein the gene isa fungal gene.
 11. A method as claimed in claim 5, wherein the viablecells in step (a) have grown into early stationary phase.
 12. A methodas claimed in claim 5, wherein the gene is a bacterial gene.
 13. Amethod as claimed in claim 5, wherein the gene is a fungal gene.
 14. Amethod as claimed in claim 6, wherein the viable cells in step (a) havegrown into early stationary phase.
 15. A method as claimed in claim 6,wherein the gene is a bacterial gene.
 16. A method as claimed in claim6, wherein the gene is a fungal gene.
 17. A method as claimed in claim6, wherein the modulatory effect is determined via optical densitymeasurements.
 18. A method as claimed in claim 7, wherein the viablecells in step (a) have grown into early stationary phase.
 19. A methodas claimed in claim 7, wherein the gene is a bacterial gene.
 20. Amethod as claimed in claim 7, wherein the gene is a fungal gene.
 21. Amethod as claimed in claim 7, wherein the modulatory effect isdetermined via optical density measurements.
 22. A method as claimed inclaim 8, wherein the viable cells have grown into early stationaryphase.
 23. A method as claimed in claim 8, wherein the gene is abacterial gene.
 24. A method as claimed in claim 8, wherein the gene isa fungal gene.
 25. A method as claimed in claim 8, wherein themodulatory effect is determined via optical density measurements.